Human cytomegalovirus (HCMV) is a frequent opportunistic pathogen, causing extensive morbidity and mortality. It is the most common known congenital virus infection in the United States, sometimes with serious or fatal consequences. In immunocompromised individuals HCMV infection or reactivation can be fatal. HCMV is a particular problem in AIDS patients, and may be a cofactor for HIV pathogenesis. A better understanding of the molecular mechanisms of HCMV DNA replication and their regulation is needed to aid in developing effective antiviral strategies. Lytic-phase DNA replication requires both a cis-acting origin of DNA replication, oriLyt, and transacting factors such as the virus-specified DNA polymerase. Eleven distinct HCMV loci, plus oriLyt, were implicated in origin-mediated DNA replication by transient complementation experiments using subclones of the viral genome. In addition, the open reading frame UL59, which lies within oriLyt, might also be required. The specific aims of this proposal are: (i) to determine if UL59 is required to complement DNA synthesis; (ii) to establish whether some of the loci that are required in trans for transient complementation are needed solely to allow expression of essential replication proteins, or if all loci encode proteins that participate directly in initiating or performing DNA replication; and (iii) to overexpress, purify, and characterize candidate replication proteins UL84, UL36, UL37, UL37ex1, and IRS1/TRS1, and to prepare immunological and biochemical tools for detailed analysis of their possible roles in DNA synthesis. These experiments will focus especially Ion those proteins that most likely play a role in regulating and promoting initiation. The methods to be used include: (i) a transient complementation assay using heterologous promoter constructs designed to allow expression independent of HCMV-specified transactivators; (ii) prokaryotic and eukaryotic systems for protein overexpression; and (iii) standard biochemical techniques including electrophoresis and column and affinity chromatography to purify and characterize the protein factors. The long term goals are to contribute to a comprehensive understanding of the molecular mechanisms and biological strategies of HCMV DNA replication and to the development of an in vitro DNA replication system, and to aid development of novel antiviral agents.